The long term goals of the proposed program are to investigate and characterize the biochemistry of trehalose formation and utilization in methylotrophic yeast. The immediate short term aims of the current proposal are to obtain fundamental basic chemical and biochemical information regarding the above processes as a prelude to further study. They include determining precise quantities of trehalose synthesized by the methylotrophic yeast during different conditions of growth and quantitation of the time course of these changes when cells are shifted between different growth conditions. The activities of the major enzymes involved in trehalose synthesis and degradation will measured. Finally these enzymes will be purified and characterized and their modes of regulation investigated. Trehalose is a non-reducing disaccharide composed of two glucopyranose units in [alpha-l,alpha-1] glycosidic linkage. It is of acute significance to human health since the dimycolate ester of this sugar has been postulated to play a critical role in the pathogenicity of bacteria that cause tuberculosis. Its function in many systems is that of a reserve energy source, particularly during sporulation or differentiation. and its levels in cells can vary depending upon the stage of development, growth conditions or environments. In bakers' yeast it is synthesized in response to starvation and heat shock and also protects against desiccation, whereas in bacteria it plays critical role as an osmoprotectant, and is rapidly synthesized when cells are subjected to high osmolarity. Its synthesis proceeds in two steps from glucose-6-phosphate and UDP-glucose to trehalose-6-phosphate which is then hydrolyzed to free trehalose. For mobilization trehalose is cleaved to two glucose residues by the action of trehalase. It is proposed to study in more detail the biochemistry of these processes in the methylotrophic yeast about which nothing is currently known. Specifically the amounts of trehalose produced by the yeast when grown on glucose/NH3, methanol/NH3 and glucose/CH3NH2 will be determined and the time courses of trehalose synthesis and hydrolysis when cells are changed between these conditions will be measured. The activities of the enzymes trehalose-6-phosphate synthase, trehalose-6-phosphate phosphatase and trehalase will be assayed during each of the above conditions as will the kinetics of changes in activity observed following changes in growth conditions. The enzymes will be isolated, purified -and characterized using advanced chromatographic and electrophoretic techniques. The possible regulation of the activities of these enzymes by covalent modification will also be analyzed, using activity measurements and electrophoretic analyses.